CN106198817B - The online separator and application method of protein N-terminal peptide fragment - Google Patents
The online separator and application method of protein N-terminal peptide fragment Download PDFInfo
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- CN106198817B CN106198817B CN201610491898.6A CN201610491898A CN106198817B CN 106198817 B CN106198817 B CN 106198817B CN 201610491898 A CN201610491898 A CN 201610491898A CN 106198817 B CN106198817 B CN 106198817B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/065—Preparation using different phases to separate parts of sample
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract
The invention belongs to bioanalysis and detection technique field, the online separator and application method of specially a kind of protein N-terminal peptide fragment.The device includes: two high-pressure pumps, six-way valve, four-way valve, triple valve, C18 column and sulfydryl peptide fragment trapping column and associated connecting member.In the present invention, proteinaceous solid is scheduled in the capillary column of filling C18 hydrophobic filler, and then carries out amino closing on column, is digested on column, and peptide hydrolysis solid phase sulfydryl is derivative, and the non-N-terminal peptide fragment of sulfydryl peptide fragment trapping column trapping realizes the online separation of N-terminal peptide fragment.The protein blocking reaction of solid phase can reduce the loss in desalting process, simplify experimental procedure, shorten the example reaction time；Meanwhile it can realize that the efficiently quick sulfydryl of peptide fragment is derivative by using excessive derivative reagent.The present invention is by on-line equipment, so that the reaction time shortens, reduces sample transfer, reduces sample loss, shorten the time-consuming of the pre-treatment steps such as desalination, improve protein N-terminal peptide fragment identification result.
Description
Technical field
The invention belongs to bioanalysis and detection technique field, and in particular to a kind of protein N-terminal peptide fragment divides online
From device and application method.
Background technique
Design combination on-line equipment, reduces pollution and loss of the sample in transfer process, in proteomics research
It is widely applied, for example the research applied to Phosphorylated Peptide reduces the absorption of sample in transfer process by direct loading,
So that the Phosphorylated Peptide number finally identified is about improved to original 4 times；By constructing in line platform, with off-line method phase
Than that can shorten time for sample pretreatment, the albumen initial amount of sample can be further decreased, be applied to the egg of a small amount of cell
In white identification.
Identify protein N-terminal sequence and its posttranslational modification, to the protein sequence of corrective gene group translation, simultaneously
Explore relevant protein function.And in current experiment route, offline mode is mostly used, needs to restore sample
It is alkylated, the amino of albumen level is closed, digested etc., it is related to multiple exchange of solvent and sample transfer；Meanwhile experimentation consumes
Duration, the problems such as be easy to causeing the albumen in sample to degrade.
Considered based on the above two o'clock, the present invention realizes albumen by the online separator of design protein N-terminal peptide fragment
Matter N-terminal efficiently separating and identifying.Firstly, proteopexy is carried out the amino closing of solid phase, which can pass through increasing
The concentration of closed reagent reduces the reaction time；Meanwhile the albumen of immobilization, it is extra to remove simply by the mode of cleaning
Salinity, reduce the loss of sample transfer process and exchange of solvent；In conjunction with immobilization proteinase solution etc., protein N is erected
Terminal peptide fragment in wire separation system so that N-terminal peptide fragment identification number further increases.
Summary of the invention
The purpose of the present invention is to provide a kind of online separator of protein N-terminal peptide fragment and application methods.
The online separator of protein N-terminal peptide fragment provided by the invention, the device include: two high-pressure pumps, one
Six-way valve, a four-way valve, a triple valve, a C18 column and a sulfydryl peptide fragment trapping column and associated connecting member.Its
In, six-way valve has 6 interfaces, and high-pressure pump 1 is connected with No. 6 mouths of six-way valve, and No. 3 mouths of six-way valve are injection port, and injection annulus connects
It is connected between No. 1 mouth of six-way valve and No. 4 mouths, C18 column is connected between No. 5 mouths of six-way valve and No. 2 mouths of four-way valve, and six is logical
No. 3 mouths of valve connect waste liquid bottle with No. 1 mouth of four-way valve, and high-pressure pump 2, No. 3 mouths of four-way valve and sulfydryl peptide fragment trapping column are used
Triple valve is connected.Wherein, high-pressure pump 1 has A, B, C and D totally 4 phases, corresponds respectively to 4 phases of A, B, C and D: A phase be from
Sub- water；B phase is amino closed reagent, is formed by formaldehyde and sodium cyanoborohydride Fresh, and concentration of formaldehyde is 0.2-3 M, cyanogen
Base sodium borohydride concentration is 0.1-1.5 M；C phase is triethyl ammonium bicarbonate solution, and concentration is 20 mM-1 M；D phase is containing 0.1%
The acetonitrile of trifluoroacetic acid.As shown in Figure 1.
In the present invention, proteinaceous solid is scheduled in the capillary column of filling C18 hydrophobic filler, and then carries out amino envelope on column
It closes, is digested on column, peptide hydrolysis solid phase sulfydryl is derivative, and sulfydryl peptide fragment trapping column traps non-N-terminal peptide fragment, realizes N-terminal peptide fragment
Online separation.
The present invention realizes sample injection of proteins using the switching of six-way valve, digests and the sulfydryl of peptide hydrolysis is derivative；Utilize dress
It is filled with the sulfydryl peptide fragment trapping column of gold modification filler, realizes the online trapping of the peptide fragment containing sulfydryl.In the separation process, solid phase
Protein blocking reaction can reduce the loss in desalting process, simplify experimental procedure, shorten the example reaction time；Meanwhile solid phase
Derivatization reaction in, can be by using excessive derivative reagent, realizing peptide fragment, efficiently quick sulfydryl is derivative.The present invention passes through
Online makeup is set, so that the reaction time shortens, is reduced sample transfer, is reduced sample loss, shorten the pre-treatment steps such as desalination
Time-consuming can be convenient and efficiently remove non-N-terminal peptide fragment, improve protein N-terminal peptide fragment identification result.
The application method of the online separator of protein N-terminal peptide fragment provided by the invention, basic step are as follows:
(1) six-way valve and four-way valve are held at A, protein sample is injected into injection annulus with sample introduction needle；
(2) six-way valve is switched to B, is passed through the sample in injection annulus with the flow velocity of 0.01-0.4 μ L/min with A phase
C18 column；
(3) six-way valve is switched to A, with the flow velocity of 0.01-0.4 μ L/min, B phase is successively passed through into C18 column, reacted
0.2-4 hours, it is then passed through A phase, C18 column is rinsed and trypsin solution is simultaneously injected into injection annulus with sample introduction needle；
(4) six-way valve is switched to B, with C phase by the trypsin solution in injection annulus with 0.01-0.4 μ L/min's
Flow velocity passes through C18 column；
(5) six-way valve is switched to A, Te Laote reagent injector is entered into injection annulus with sample introduction needle, meanwhile, it is rinsed with A phase
C18 column；
(6) six-way valve is switched to B, with C phase by the Te Laote reagent in injection annulus with the stream of 0.01-0.4 μ L/min
Speed passes through C18 column；
(7) six-way valve is switched to A, four-way valve is switched to B, washed with the flow velocity of 0.01-0.4 μ L/min with D phase
De- C18 column, meanwhile, pump 2 is passed through A phase with 0.2-4 times of flow velocity, and mixed liquor enters sulfydryl peptide fragment trapping column, and efflux enters liquid phase
Chromatograph-mass spectrometer is detected.
Above step is as shown in Figure 2.
In the present invention, C18 column the preparation method is as follows: disperse C18 filler in acetonitrile, using air pressure, filler is filled out
Enter in capillary.Sulfydryl peptide fragment trapping column the preparation method is as follows: firstly, trifluoro ethanesulfonic acid based high-polymer material and half will be contained
Cystine mixing, is placed in the buffer system of alkalinity and is incubated for；Second, the polymeric material of cysteine modified is obtained by centrifugation
Material；After washing several times, the material and pre-synthesis nano Au colloid are incubated for, centrifugation obtains the height of decorated by nano-gold
Polymers filler；The filler is dried, and is scattered in dehydrated alcohol by third, is inserted filler in capillary using air pressure.
Using the online separator for the protein N-terminal peptide fragment that the present invention designs, can be completed in 3 hours all real
Test step, comprising: amino closing, enzymatic hydrolysis, sulfydryl derivative and the trapping of sulfydryl peptide fragment of albumen etc. have comparable application potential.
Detailed description of the invention
Fig. 1 is integrated apparatus figure of the invention.
Fig. 2 is the online separation process figure of N-terminal peptide fragment.Wherein, A phase is deionized water；B phase is amino closed reagent, by
Formaldehyde and sodium cyanoborohydride Fresh form, and concentration of formaldehyde is 0.2-3 M, and sodium cyanoborohydride concentration is 0.1-1.5 M；
C phase is triethyl ammonium bicarbonate solution, and concentration is 20 mM-1 M；D phase is the acetonitrile containing 0.1% trifluoroacetic acid.
Specific embodiment
Embodiment 1: protein on a cl 8 column close and digest by amino
10 μ g are taken to be supported in C18 column through six-way valve switching the bovine serum albumin(BSA) after reductive alkylation；By six-way valve
A are switched to, pumps 1 B phase with 2 μ L/min by C18 column, after reaction 1 hour；Then, then with A phase C18 pillar is cleaned；Together
When, 30 μ L trypsin solutions are injected in injection annulus, are pumped 1 C phase and are pushed the enzyme in injection annulus molten with the flow velocity of 2 μ L/min
Liquid passes through C18 column；After reacting half an hour, the peptide fragment on C18 column is eluted using C phase, utilization is substance assistant laser desorpted ionized
Flight time mass spectrum is identified.The result shows that the amino of bovine serum albumin(BSA) energy can be closed successfully, therewith by effectively enzyme
Solution.
The derivative trapping effect with sulfydryl peptide fragment trapping column of sulfydryl on embodiment 2:C18 column
After acetylation peptide fragment GR(1021.5 Da) and the free peptide fragment ER(1282.6 Da of amino) injection C18 column, then into
120 μ L of sample, 2 M Te Laote reagent, with the C phase of pump 1 by the reagent with 2 μ L/min by C18 column, then, then with D phase by mercapto
Peptide fragment elution after base is derivative.At this point, four-way valve is switched to B, pump 2 injects deionized water with identical flow velocity, elutes and goes
Ionized water enters sulfydryl peptide fragment trapping column through triple valve.It identifies, flows out through Matrix-assisted laser desorption ionization
Only detect the signal of peptide fragment GR in liquid, it was demonstrated that sulfydryl derivative works well on C18 pillar, and peptide fragment containing sulfydryl is by successfully
Trapping, N-terminal peptide fragment can be separated efficiently.
Claims (3)
1. a kind of online separator of protein N-terminal peptide fragment characterized by comprising two high-pressure pumps, one six it is logical
Valve, a four-way valve, a triple valve, a C18 column and a sulfydryl peptide fragment trapping column and associated connecting member；Wherein,
Six-way valve has 6 interfaces, and the first high-pressure pump is connected with No. 6 mouths of six-way valve, and No. 3 mouths of six-way valve are injection port, and injection annulus connects
It is connected between No. 1 mouth of six-way valve and No. 4 mouths, C18 column is connected between No. 5 mouths of six-way valve and No. 2 mouths of four-way valve, and six is logical
No. 2 mouths of valve connect waste liquid bottle, the second high-pressure pump, No. 3 mouths of four-way valve and sulfydryl peptide fragment trapping column with No. 1 mouth of four-way valve
It is connected with triple valve；Wherein, the first high-pressure pump has A, B, C and D totally 4 phases, corresponds respectively to 4 phases of A, B, C and D: A phase
For deionized water；B phase is amino closed reagent, is formulated by formaldehyde and sodium cyanoborohydride, and concentration of formaldehyde is 0.2-3 M,
Sodium cyanoborohydride concentration is 0.1-1.5 M；C phase is triethyl ammonium bicarbonate solution, and concentration is 20 mM-1 M；D phase be containing
The acetonitrile of 0.1% trifluoroacetic acid.
2. the online separator of protein N-terminal peptide fragment according to claim 1, which is characterized in that protein is fixed
In the capillary column of filling C18 hydrophobic filler, and then amino closing on column is carried out, is digested on column, peptide hydrolysis solid phase mercapto
Base is derivative, and sulfydryl peptide fragment trapping column traps non-N-terminal peptide fragment, realizes the online separation of N-terminal peptide fragment.
3. the application method of the online separator of the protein N-terminal peptide fragment according to claim 1 or 2,
Be characterized in that specific steps are as follows:
(1) six-way valve and four-way valve are held at A, protein sample is injected into injection annulus with sample introduction needle；
(2) six-way valve is switched to B, the sample in injection annulus is passed through into C18 with the flow velocity of 0.01-0.4 μ L/min with A phase
Column；
(3) six-way valve is switched to A, with the flow velocity of 0.01-0.4 μ L/min, B phase is passed through into C18 column, reaction 0.2-4 is small
When, it is then passed through A phase, C18 column is rinsed and trypsin solution is injected into injection annulus with sample introduction needle at the same time；
(4) six-way valve is switched to B, with C phase by the trypsin solution in injection annulus with the flow velocity of 0.01-0.4 μ L/min
Pass through C18 column；
(5) six-way valve is switched to A, Te Laote reagent injector is entered into injection annulus with sample introduction needle, meanwhile, C18 is rinsed with A phase
Column；
(6) six-way valve is switched to B, is led to the Te Laote reagent in injection annulus with the flow velocity of 0.01-0.4 μ L/min with C phase
Cross C18 column；
(7) six-way valve is switched to A, four-way valve is switched to B, C18 is eluted with D phase with the flow velocity of 0.01-0.4 μ L/min
Column, meanwhile, the second high-pressure pump is passed through A phase with 0.2-4 times of flow velocity, and mixed liquor enters sulfydryl peptide fragment trapping column, and efflux enters liquid
Phase chromatograph-mass spectrometer is detected.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610491898.6A CN106198817B (en) | 2016-06-29 | 2016-06-29 | The online separator and application method of protein N-terminal peptide fragment |
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| CN201610491898.6A CN106198817B (en) | 2016-06-29 | 2016-06-29 | The online separator and application method of protein N-terminal peptide fragment |
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| CN106198817B true CN106198817B (en) | 2019-01-15 |
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| CN109060992B (en) * | 2018-09-03 | 2024-02-27 | 苏州博睿嘉晟医疗科技有限公司 | Glycosylated hemoglobin analyzer capable of improving detection accuracy |
| CN110220999A (en) * | 2019-07-10 | 2019-09-10 | 中国南方电网有限责任公司超高压输电公司检修试验中心 | Chromatographic column automatic back blow sweeps activation device in a kind of transformer oil |
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| CN105319116B (en) * | 2015-11-18 | 2018-11-13 | 复旦大学 | Protein N-terminal peptides separation method based on magnetic microsphere |
| CN105316381B (en) * | 2015-11-18 | 2019-06-21 | 复旦大学 | The method that the graphene of decorated by nano-gold is used for protein N-terminal separation |
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